PROJECT SUMMARY High grade gliomas in both adults and children confer very poor prognosis, with median survival rates under two years post diagnosis. Amplification of epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), as well as mutations in the PI3K/AKT/mTOR pathway are frequent and can be targeted by kinase inhibitor based strategies. EGFR inhibition has been tested clinically, but responses have been limited. Inadequate target engagement, perhaps due to poor blood brain barrier penetrance, as well as downregulation or circumvention of the target in recurrent tumors, are amongst mechanisms cited for lack of efficacy of EGFR-targeted agents. Combination strategies using PI3K and MAPK inhibitors are promising, and can overcome acquired resistance to single agents in GBM. Advantages of using a single molecule to target multiple kinases include reduced risks of drug interactions, a single pharmacokinetic profile for optimization of dosing, and increased potential for overcoming drug resistance. Therefore, the development of therapeutics that target more than one kinase, identification of biomarkers of response and careful evaluation of BBB permeance are imperative to improving patient outcomes. Employing a computational modeling approach, we exploited the known binding modes of structurally related ATP binding site inhibitors of EGFR and PI3K to design small molecules that simultaneously inhibit both kinases in a selective manner. We hypothesize that this polypharmacology approach will provide better efficacy in vitro and in vivo compared to multi-drug combination strategies. Preliminary data generated in human glioblastoma and patient derived pediatric diffuse intrinsic pontine glioma (DIPG) lines shows potent cytotoxic effects of these dually targeted agents relative to targeting of EGFR alone or PI3K alone. Furthermore, we have identified unique metabolic features of the inhibitors, indicating suppression of both glycolytic pathways and oxidative phosphorylation, which is not seen with clinically relevant EGFR or PI3K inhibitors. We posit that this will lead to the development of metabolomics based biomarkers. In Year 1, in the R61 phase of this application, we will further optimize the pharmaceutical features of this chemical series of compounds to increase the likelihood of blood-brain barrier (BBB) penetrance. We have generated key preliminary data showing that MTX-241 possess critical attributes for BBB permeability. Therefore we will use in vivo models to test MTX-241, while simultaneously synthesizing and evaluating 15-20 structural analogs of MTX-241. The R33 phase of the proposal will be carried out in Years 2 and 3, whereupon we will focus on in vivo evaluation of the two most promising candidates identified using orthotopic models to address anti-tumor efficacy, blood brain barrier permeance, and biomarkers of drug action. These studies have been designed to support our hypothesis that a single inhibitor with dual specificity for EGFR and PI3K represents a rationale and promising treatment strategy for recurrent adult and pediatric HGG with high translational potential.